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Kupfer-type Immunological Synapses in vivo: Raison D’être of SMAC Izaskun Mitxitorena, Elena Saavedra, Carlos Barcia, Ph.D. Department of Biochemistry and Molecular Biology, Institute of Neuroscience & School of Medicine, Universitat Autonoma de Barcelona, Lab M2-107, Bellaterra, Cerdanyola del Valles, Barcelona, Spain. Running Title: SMAC formation in T cells and therapeutic perspectives Key words: Immunological Synapses, Supramolecular Activation Cluster, Glioma, viral infection, and immunotherapy Corresponding Author: Carlos Barcia Institute of Neuroscience

Department of Biochemistry and Molecular Biology School of Medicine, Lab M2-107 Autonomous University of Barcelona Cerdanyola del Vallès, 08193, Barcelona, Spain carlos.barcia@uab.es

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Abstract

T cells engage with antigen-presenting cells to form immunological synapses. These

intimate contacts are characterized by the complex arrangement of molecules at the

intercellular interface, which has been described as the supramolecular activation cluster

(SMAC). However, due to T cells functioning without SMAC formation and the

difficulties of studying these complex arrangements in vivo, its biological importance has

been questioned. In light of recent data, we focus this review on the putative functionality

of SMACs in T-cell synaptic contacts in vivo and emphasize the therapeutic potential of

SMAC manipulation in immune-driven diseases.

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Immunological Synapse Formation and SMAC arrangement

Immunological synapses (IS) are critical intercellular communications between specific

immune cells and antigen-presenting cells (APC)1. This particular engagement between

both counterparts requires intimate contact between the aforementioned cells and

includes multiple factors and complex signaling cascades of activation 1,2. T-cell ISs have

been largely studied and represent the best-known IS type 3, although ISs may also be

established by different types of effector cells, such as NK or B cells 4-6. The formation of

an IS involves the T-cell recognition of specific antigens that are presented by APCs.

Major Histocompatibility Complexes (MHC) display antigens at the APC cell surface,

which are detected by T-cell receptor (TCR) molecules that are displayed on the T-cell

membrane 7. The interaction between the antigen-MHC and the TCR induces the TCR

signaling cascade 8, thus initiating the activation of the T cell, which is characterized by

the phosphorylation and polarization of tyrosine kinases such as lymphocyte-specific

protein tyrosine kinase (Lck) and zeta-chain-associated protein kinase 70 (ZAP-70) at the

interface 9,10 (Figure 1). In mature IS formation, the process of activation involves severe

changes to the micro-anatomical configuration of the T cell that are characterized by

rearrangement of the actin cytoskeleton and are driven by the microtubules organizer

center (MTOC), which becomes polarized toward the APC and participates in the

organization of secretory domains 11-14. The polarization of the T cell is also accompanied

by the rearrangement of lymphocyte function-associated antigen 1 (LFA-1) molecules

that segregate three-dimensionally at the IS interface and specifically bind to the APC’s

intercellular adhesion molecule 1 (ICAM-1) 15,16. This binding of LFA-1/ICAM-1 takes

place at the interface, and LFA-1/ICAM-1 complexes rearrange micro-anatomically,

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forming a ring-shaped area named the peripheral supramolecular activation cluster

(pSMAC), which surrounds a characteristic central accumulation of TCRs, known as the

central supramolecular activation cluster (cSMAC) 15 (Figure 1 and Box). This way, a

“bull’s eye” characteristic structure is formed, where an outer ring contains the adhesion

molecules, and an inner area contains the signaling molecules. In cytolytic T cells, the

cSMAC may also contain secretory domains that usually encompass an area of smaller

size and is located near the TCR signaling central cluster, where lytic granules of effector

molecules are concentrated and released 6,17,18. Importantly, LFA-1 molecules are linked

to talin proteins, which are key integrins involved in cell migration and cellular junction

because they are linked to the actin-myosin cytoskeleton through vinculin 15,19,20.

Visualization of SMACs in vivo

The initial description and most of the studies on the microanatomy and function of ISs

have been performed in vitro 1,15,21. Although the knowledge on ISs has substantially

grown and successfully improved based on in vitro experiments, the functionality of ISs

in living organisms has barely been explored. A criticism often rises considering that in

vitro environments are different from those in tissue. Cultures and planar bilayers are

isolated, two-dimensional milieus, whereas tissues are three-dimensional environments in

which cells receive information and signals from different planes and directions

involving diverse biological systems. Thus, research of ISs in vivo is an important matter

for a complete understanding of T-cell biology.

Formation of SMAC in vivo has been demonstrated using high-resolution

confocal microscopy of labeled, fixed tissue with multiple fluorescence-specific

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antibodies. The formation of the CD3/TCR central cluster (cSMAC) and/or the peripheral

segregation of LFA-1 (pSMAC) are observed in different tissues, such as the brain and

secondary lymphoid tissues 22,23. ISs are stable and preserved structures in mammals. As

described in vitro, ISs show a flat interface in vivo; and cSMAC and pSMAC are formed

in all species studied so far. From rodents 22 to primates 24, including humans 25, the

formation of SMAC seems to be consistently involved in mammalian immune responses.

However, despite the good level of resolution, this in vivo technique has the

limitation of picturing static events. High-resolution confocal images in fixed tissue

represent a scenery taken at a certain and specific moment and do not resolve the

dynamics of the IS. Two-photon microscopy in living animals will be the ideal technical

approach to show the dynamics of IS formation in vivo, but some issues must still be

solved. Currently, multi-photon microscopes are able to image several hundreds of

microns deep into tissue; however, the resolution of the anatomical details is still not

sufficient to distinguish the micro-anatomy of the IS at the SMAC level. In addition,

observations are hampered by the parenchyma’s high auto-fluorescence and by the

reduced number of fluorophores that are available to detect molecule arrangements in

vivo in time-lapse, live imaging. Two-photon microscopy studies in tissue, especially in

lymph nodes, have shown the dynamics by which T cells engage APCs (i.e., dendritic

cells), but no micro-anatomical details of the SMAC were given 26,27. Currently, time-

lapse studies of the microanatomy of complete SMAC formation, containing the central

and peripheral clusters, have not been yet performed in living tissue. Notably, however, a

successful attempt was performed regarding visualization of the dynamics of the

formation of the TCR central cluster using a two-photon microscope in lymph nodes in

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live mice. In a study by Friedman et al., some features of the TCR dynamics in vivo, as

well as the behavior of TCR accumulation, were revealed 28. In addition Azar et al., using

linker for activation of T cells (LAT)-EGFP labeled T cells, were able to detect the in

vivo formation of central and peripheral clusters of LAT at the IS interface in lymph

nodes, which may underlie some insights into the molecular distribution of SMACs 29.

The next scientific challenge is the combination of different fluorophores to observe the

dynamics of the peripheral SMAC in relation to the central TCR cluster and how the

formation of these structures affects immune responses in healthy subjects and

experimental models of diseases.

Function of SMAC in vivo

Previous observations have shown that SMAC formation is not required for TCR

signaling or for the effectiveness of cytotoxic T cells 6,30. These results question the

biological importance of SMAC formation. Why is such an enormous and complex

arrangement in the cell needed? Why invest such a large amount of energy and effort?

pSMAC and cSMAC formations were first observed in brain tissue, in the context of the

clearance of virus-infected cells 22. In this case, the formation of SMACs preceded the

elimination of viral-infected cells in immune-competent animals that were primed with

an adaptive immune response 22. In this context, the percentage of ISs forming SMACs

and engaged with virus-infected cells was approximately 60% in a specific time window,

before complete viral clearance 31,32. These results indicate that a large percentage of

SMAC formation may be essential for viral clearance in tissue, suggesting its biological

significance 31. In the same scenario of viral clearance, the secretory domain that was

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observed at the immunological synaptic interface was characterized by the formation of

interferon-gamma (IFN- ) and perforin clusters, which conveys that both effector

molecules and their polarization at the synaptic interface may be necessary phenomena

for the elimination of virus-infected cells 31. In fact, IFN- - or perforin-deficient mice are

unable to eliminate virus-infected cells from the brain 33. However, whether completely

mature SMAC rearrangements will take place at the interface seems to depend upon

multiple factors. For example, IFN- appears polarized in Kupfer type (with SMAC) and

non-Kupfer type (without SMAC) synapses 31, which indicates that the formation of

mature synapses with SMAC does not precede the formation of the secretory domain;

therefore, SMAC formation may not be strictly necessary for the release of effector

molecules and elimination of target cells. In fact, although cytotoxic ISs restrict killing to

antigenic target cells, IFN- signaling is also detected in non-antigenic bystander cells 34,

suggesting a certain leakage or multidirectional diffusion of the cytokine, which implies

defective SMAC formation.

On the other hand, secretory effector molecules have a different pattern of

segregation that is independent of c- and pSMAC formation. Therefore, different

cytokines show different patterns of secretion in T cells. For example, IFN- and

interleukin 2 (IL-2) are polarized and secreted to the synaptic interface, while TNF- and

chemokine (C-C motif) ligand 3 (CCL3) are secreted multi-directionally 35. These

established patterns of secretion indicate a different behavior of T-cells that depends on

the context of the immune response. Thus, the need for complex SMAC rearrangement

may not always be required.

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These results indicate that SMAC arrangement could be necessary to directionally

secrete specific molecules towards the APC without altering adjacent cells, thus safely

channeling intercellular communication 13,36 (Figure 2). Outer ring LFA-1/ICAM-1

adhesion allows for the formation of a shielded micro-chamber, which is an intercellular

space that is kept isolated from the surrounding environment. This flat interface feature is

possible due to rearrangement of the actin cytoskeleton, which forms a consistent and

renewable scaffold that is oriented to the interface 37,38. Most likely, the reason for these

interface arrangements may be for maximal reduction of the surface at the intercellular

contact, which could result in more effective communication and less chance of

membrane and receptor miss-folding. In that intercellular space, cytotoxic compounds,

such as effector molecules, can be safely delivered, and signaling only occurs with the

contacting cell, without damaging the surrounding healthy cells that are not involved in

the immunological response. Therefore, the formation of SMACs may represent a highly

evolved and specific immune response that only has an effect on target cells and does not

affect bystander cells. Thus, it can be hypothesized that the SMAC is a necessary

structure to channel cytokines and other effector molecules in an extremely selective

manner (Figure 2).

Overall, T-cell synaptic contacts may be necessary for an effective immune

response, but, the formation of SMACs may depend on the immunological context and

the effector molecules that are delivered. It is, therefore, tempting to speculate that the

ideal situation may be SMAC formation because it would preserve the surrounding tissue

and result in a more specific and safe response. As a drawback, an immune response with

SMAC formation is most likely slower and requires high-energy waste. Thus, if the

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immune response needs to be faster and inexpensive, it should be carried out without

SMAC.

In summary, in vivo studies of Kupfer-type ISs exhibit a complex scenario for

further research. Multiple types of intercellular combinations, involving diverse cytokine

release and adaptable immune responses within different tissues, are important variables

that should be considered for future research, although the visualization and unraveling of

the IS function will only be fully achieved in vivo if new, specific approaches are

designed that selectively inhibit IS formation in the tissue of a living organism.

A therapeutic view of the immunological synapse

Because the formation of the SMAC may be an important part of the specificity and

effectiveness of the T-cell response, manipulation of ISs represents a promising tool from

a therapeutic point of view. It presents an advantage whereby we could specifically

inhibit or activate the different immune responses according to therapeutic needs, as

multiple targets could potentially be aimed to hinder or empower IS formation. In fact,

immunotherapy is a therapeutic field that has lately been developed and is becoming

promising, particularly for cancer. Specific drugs, usually artificially made antibodies,

have been designed to empower anti-tumor immunity, and most of them intervene at the

synaptic level (Figure 3).

One of the most hopeful approaches to directly stimulate the formation of specific

ISs between T and tumor cells is the development of bi-specific T-cell engager (BiTE)

antibodies. These monoclonal antibodies target the TCR/CD3 complex and tumor

antigens, such as CD19, epithelial cell adhesion molecule (EpCAM) or epidermal growth

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factor receptor (EGFR). This way, the antibodies promote the synaptic interaction

between tumor cells and T cells and induce the activation of cytolytic T cells. This

engagement-induced tumor-cell death leads to T-cell accumulation in the tumor

microenvironment and reduces tumor cell proliferation in vivo 39.

Another successful approach to modify synaptic contacts is based on the

development of antibodies that are able to antagonize receptors that inhibit the immune

response. A successful case is that of ipilimumab, an antibody that binds an inhibitory T-

cell protein called cytotoxic T lymphocyte antigen 4 (CTLA-4). CTLA-4 is expressed in

activated T cells and is recruited to the cSMAC in competition with the T-cell activation

molecule, CD28 40,41. The binding of ipilimumab interferes with CTLA-4-mediated T-cell

suppression at the cSMAC, therefore, facilitating active synaptic interactions between T

cells and target cells, which results in a more aggressive immune response against the

tumor. Ipilimumab has been tested in patients with melanoma (Yervoy®), and it has been

proven to be effective in specific cases because it removes melanoma without tumor

recurrence 42-44. Analogously, therapeutic blockade of programmed cell death 1 (PD-1),

which is also localized at the cSMAC, increases T-cell motility and cytotoxic

effectiveness, thus improving viral clearance 45. Indeed, the combination of both, CTLA-

4 and PD-1 blockade, has been proven to be effective toward tumors by increasing the

cytolytic T-cell population and reducing regulatory T cells 46.

In this context, optimization of the cytolytic arm seems to be the primary therapeutic

strategy to eliminate tumors because the tumorigenic microenvironment facilitates a pro-

inflammatory response that promotes tumor growth. In the case of CNS tumors,

particularly in human glioma, the formation of SMAC has been studied in depth. In

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glioma tissue, mature ISs are established between T-cells and tumorigenic cells, although

at a low rate 25. However, SMAC analyses performed in murine experimental models of

glioma have shown that the formation of Kupfer-type synapses does not predict the

elimination of the tumor 47, which is different from the process of viral clearance 31. This

feature may be characteristic of tumors because the multidirectional delivery of cytotoxic

compounds could theoretically be the fastest and most effective way to destroy tumors in

an environment where the majority of bystander cells should be rapidly eliminated.

However, because T cells form SMACs, they may still be needed in a sufficient quantity

for the recognition of specific antigens to take place. This fact supports the idea that

SMACs would only be formed when the tissue in the vicinity must be preserved. These

concepts may open new avenues of research regarding the formation of SMAC or

bonafide ISs.

On the other hand, tumor development and other immune-mediated degenerative

diseases might be a consequence of defective SMAC formation. This alteration may be

reflected in altered immune responses due to deficient recognition of the antigen, anergy

or exhaustion of the T-cell response, either of the regulatory or cytolytic response. In line

with this, a recent study showed for the first time that alterations in SMAC formation in T

cells can be a crucial element in immune disorders. In this report, CD4 T cells obtained

from patients with multiple sclerosis and type-1 diabetes were exposed to antigens from

influenza virus. Both CD4-T-cell groups showed divergent formation of SMAC when

compared with normal T cells obtained from healthy patients 48. These differences

included deficient SMAC-structure formation regarding the proper CD3/TCR or MHC

accumulation and ICAM-1/LFA-1 segregation, a distinct motility of T cells, and altered

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timing and velocity of SMAC formation. Importantly, a deficiency in SMAC formation

sets the possibility for alteration in cellular communication and could explain how T cells

might escape the negative selection that takes place in autoimmune diseases.

Another example regarding the X-linked lymphoproliferative syndrome, which is

characterized by fatal responses to Epstein-Barr virus infection, has recently been

reported. This syndrome is caused by mutations affecting the adaptor SAP (signaling

lymphocytic activation molecule (SLAM)-associated membrane protein), which is a

molecule involved in correct arrangement of the synaptic contact. In fact, SAP-deficient

cytotoxic T lymphocytes exhibit abnormal actin organization and reduced centrosome

docking at T-cell–B-cell ISs 49. These results demonstrate that correct assembling of T

cells with their target cells and the micro-anatomical arrangement of SMACs and their

associated organelles is a fundamental process in the immune response.

We are beginning to understand how malfunction of SMAC formation may induce

different immune-mediated diseases. The understanding of this process in vivo as well as

the specific mechanisms occurring during SMAC formation in tissues within different

immune scenarios will be crucial to propose molecular targets that restore the correct

arrangement of Kupfer-type ISs.

Box

The term immunological synapse has been used to generally define communications

between immune cells, although it is also specifically and more accurately referred to as

the formation of the characteristic interface with complex rearrangements of molecules

and compounds called SMAC (Supra-Molecular Activation Cluster). Synapses that form

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SMACs are considered mature immunological synapses and, in some publications, to

honor its discoverer, immunological synapses are classified as Kupfer-type or non-

Kupfer-type immunological synapses according to the presence or absence of the “bull’s

eye” formation at the interface, respectively.

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Figure Legends

Figure 1. T-cell immunological synapse forming a SMAC (Kupfer-type). T cells

recognize antigens that are presented by the MHC of an APC through the TCR/CD3

complex. Then, T cells are activated through phosphorylation of tyrosine kinases such as

Lck and ZAP-70, which are polarized to the T-cell/APC interface. This activation leads

to dramatic changes in the cell, including the rearrangement of adhesion molecules, such

as LFA-1, which are segregated towards the interface to bind ICAM-1 of the APC and

form the peripheral activation cluster (pSMAC). On the other hand, TCR/CD3 molecules

are aggregated at the center of the interface and form the central SMAC (cSMAC). In

addition, cytotoxic granules are delivered to the center of the interface and form the

secretory domain.

Figure 2. Hypothetical strategies for cytolytic T-cell responses in tissue. A.

Unidirectional secretion of effector molecules after immunological synapse formation. T

cells (red) form mature immunological synapses after antigen recognition and subsequent

apposition to an APC (blue). LFA-1 adhesion molecules are segregated at the external

border of the interface (red), forming the pSMAC, whereas TCR (green) is concentrated

at the center of the interface, forming the cSMAC, where the cytolytic granules (yellow

arrow) may be delivered in one specific direction. With this strategy, the APC (blue) can

be specifically eliminated without damaging bystander cells (light brown cells). B.

Multidirectional secretion of effector molecules without bona fide synapse formation. T

cells (red) may not form mature immunological synapses after antigen recognition; thus,

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the strict apposition to antigen-presenting cells (blue) may not be necessary. LFA-1

molecules (red) do not arrange as pSMAC, and TCR does not concentrate at the center of

the interface, forming the cSMAC. Cytolytic granules (yellow arrows) may be delivered

multi-directionally. With this strategy, bystander APCs (blue) can be eliminated

discretionally.

Figure 3. Therapeutic targets at the immunological synapse. CTLA-4 competes with

CD28 for CD80/CD86. Bound CTLA4-CD80/CD86 complexes are recruited to the

cSMAC, whereas unbound CD28 is segregated to the pSMAC. PD1 molecules bind to

PDL1 and are recruited to the cSMAC. The binding of CTLA4-CD80/CD86 inhibits T-

cell activation. Thus, CTLA-4 blocking antibodies hamper binding to CD80/CD86,

which facilitates the binding of CD80/CD86 with CD28 and impedes CTL inhibition.

Similarly, PD1-blocking antibodies obstruct the inhibition of T cells at the synaptic

interface.

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Texto1: Post-print of: "Kupfer-type immunological synapses in vivo: Raison D’être of SMAC / I. Mitxitorena, E. Saavedra and C. Barcia", in Immunology and Cell Biology (Nature), 2015, vol. 93, p. 51–56; doi:10.1038/icb.2014.80